Breaks in the phosphodiester backbone of DNA and essential RNA molecules can lead to cell death if not repaired. This project aims to illuminate the mechanisms and structures of the DNA ligase and RNA ligase enzymes that rectify such breaks. Polynucleotide ligases catalyze the joining of a 5'-PO4 strand to a 3'-OH end via 3 chemical steps: (i) ligase reacts with ATP or NAD+ to form a covalent ligase-adenylate intermediate and release pyrophosphate or NMN; (ii) AMP is transferred from the ligase to the 5'-PO4 DNA or RNA strand to form a DNA/RNA-adenylate intermediate (AppDNA or AppRNA); (iii) ligase catalyzes attack by the 3'-OH of the nick on AppDNA or AppRNA to form a phosphodiester and release AMP. Our long-range goals are to understand how ligase reaction chemistry is catalyzed and how ligases recognize "damaged" DNA or RNA ends. We are approaching these problems using a eukaryotic virus-encoded DNA ligase (Chlorella virus DNA ligase), a bacterial NAD+-dependent DNA ligase (E. coli LigA), and a bacteriophage ATP-dependent RNA ligase (T4 Rnl2) as models. Rnl2 was discovered by this laboratory during the previous funding period and quickly developed into a model-of-choice for RNA repair enzymology. Rnl2 exemplifies a new and growing family of RNA ligases found in all phylogenetic domains. Our studies, which integrate biochemistry, molecular genetics, and structural biology, have revealed mechanistic principles shared by all DNA and RNA ligases, as well as the unique structural features and substrate specificities that distinguish the various branches of the polynucleotide ligase superfamily. In particular, our work indicates that progression through the sequential steps of the ligation pathway is coupled to large protein domain movements and serial remodeling of the active site. Specific aims of this proposal are: (1) To identify the functional groups of Chlorella virus DNA ligase, E. coli LigA, and T4 Rnl2 that contribute to DNA/RNA recognition and nucleotidyl transfer; (2) To biochemically define the interface between ligase-adenylate and nicked DNA/RNA substrates; and (3) To determine atomic structures of ligase-adenylate bound at a DNA/RNA nick and of ligases bound to the 5'-adenylated polynucleotide intermediate.